The overall goal of this proposed research project is to develop ribozyme technology to the point that it is close to therapeutic reality. This will be done using the hairpin ribozyme which is a small trans-acting RNA enzyme that is highly suitable for cellular RNA inactivation studies. Previous work has elucidated rules by which the hairpin ribozyme can be engineered to efficiently cleave HIV sequences and has developed an in vitro selection method through which ribozyme activity against a given target sequence can be optimized. These findings have been used to generate a ribozyme targeted to a conserved HIV pol sequence that strongly inhibits HIV-1 replication in cultured lymphocytes. The Specific Aims of the current proposal are: (1) Rigorously define the site and mechanism of antiviral action of anti-HIV hairpin ribozymes, and assess their substrate selectivity in vivo; (2) Develop and evaluate a novel class of anti-HIV targets, using new information defining substrate specificity of the hairpin ribozyme. It is expected that this work will result in (i) definitive proof that engineered hairpin ribozymes inhibit HIV replication by site- specific RNA cleavage at the targeted site, (ii) the exploration of several novel strategies for the development, intracellular activation, and metabolic regulation of highly active ribozymes, and (iii) the development and rapid testing of a number of potential new anti-HIV therapeutic agents.